Patent

1 . An isolated antibody or antigen binding fragment thereof, comprising:

(a) a light chain variable region comprising:

(i) CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO:9);

(ii) CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO:10); and

(iii) CDR-L3 comprising the amino acid sequence AAWDERLSGWV (SEQ ID NO: 11);

and

(b) a heavy chain variable region comprising:

(i) CDR-H1 comprising the amino acid sequence SSNWWS (SEQ ID NO:12);

(ii) CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 13); and

(iii) CDR-H3 comprising the amino acid sequence GSSSWYDLGPFDY (SEQ ID NO: 14).

2 . The isolated antibody or antigen binding fragment thereof of claim 1 , wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:15 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:16.

3 . An isolated antibody or antigen binding fragment thereof, comprising:

(a) a light chain variable region comprising:

(i) CDR-L1 comprising the amino acid sequence SEQ ID NO:9;

(ii) CDR-L2 comprising the amino acid sequence SEQ ID NO:10; and

(iii) CDR-L3 comprising the amino acid sequence SEQ ID NO:11; and

(b) a heavy chain variable region comprising:

(i) CDR-H1 comprising one of the amino acid sequences selected from the group consisting of SEQ ID NO:91, SEQ ID NO: 92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO: 95, SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98;

(ii) CDR-H2 comprising the amino acid sequence SEQ ID NO:13; and

(iii) CDR-H3 comprising the amino acid sequence SEQ ID NO:14.

4 . The isolated antibody or antigen binding fragment thereof of claim 3 , wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:15 and the heavy chain variable region comprises one of the amino acid sequences selected from the group consisting of SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO:105, and SEQ ID NO:106.

5 . An isolated antibody or antigen binding fragment thereof, comprising:

(a) a light chain variable region comprising:

(i) CDR-L1 comprising the amino acid sequence RASQGIRNDLG (SEQ ID NO:1);

(ii) CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO:2); and

(iii) CDR-L3 comprising the amino acid sequence LQDHDYPLT (SEQ ID NO:3); and

(b) a heavy chain variable region comprising:

(i) CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO:4);

(ii) CDR-H2 comprising the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO: 5); and

(iii) CDR-H3 comprising the amino acid sequence GFYVRNWFDP (SEQ ID NO:6).

6 . The isolated antibody or antigen binding fragment thereof of claim 5 , wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:7 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:8.

7 . An isolated antibody or antigen binding fragment thereof, comprising:

(a) a light chain variable region comprising:

(i) CDR-L1 comprising the amino acid sequence RASQGISSYLA (SEQ ID NO:17);

(ii) CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO:18); and

(iii) CDR-L3 comprising the amino acid sequence QQYYSYPFT (SEQ ID NO:19); and

(b) a heavy chain variable region comprising:

(i) CDR-H1 comprising the amino acid sequence SYAMS (SEQ ID NO:20);

(ii) CDR-H2 comprising the amino acid sequence AISSSGSSTYYADSVKG (SEQ ID NO: 21); and

(iii) CDR-H3 comprising the amino acid sequence DQGGYGYPGESWFDY (SEQ ID NO: 22).

8 . The isolated antibody or antigen binding fragment thereof of claim 7 , wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:23 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:24.

9 . An isolated antibody or antigen binding fragment thereof, comprising:

(a) a light chain variable region comprising:

(i) CDR-L1 comprising the amino acid sequence RASQSISSYLN (SEQ ID NO:25);

(ii) CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO:26); and

(iii) CDR-L3 comprising the amino acid sequence QQSYSPPWT (SEQ ID NO:27); and

(b) a heavy chain variable region comprising:

(i) CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO:28);

(ii) CDR-H2 comprising the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO: 29); and

(iii) CDR-H3 comprising the amino acid sequence AFYSYMDV (SEQ ID NO:30).

10 . The isolated antibody or antigen binding fragment thereof of claim 9 , wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:31 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:32.

11 . An isolated antibody or antigen binding fragment thereof, comprising:

(a) a light chain variable region comprising:

(i) CDR-L1 comprising the amino acid sequence RSSQSLLHSNGYNYLD (SEQ ID NO: 33);

(ii) CDR-L2 comprising the amino acid sequence LGSNRAS (SEQ ID NO:34); and

(iii) CDR-L3 comprising the amino acid sequence MQALQTPLT (SEQ ID NO:35); and

(b) a heavy chain variable region comprising:

(i) CDR-H1 comprising the amino acid sequence SSNWWS (SEQ ID NO:36);

(ii) CDR-H2 comprising the amino acid sequence EIYHSGSTNYNPSLKS (SEQ ID NO: 37); and

(iii) CDR-H3 comprising the amino acid sequence ERTILTGYYGFDY (SEQ ID NO: 38).

12 . The isolated antibody or antigen binding fragment thereof of claim 11 , wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:39 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:40.

13 . An isolated antibody or antigen binding fragment thereof, comprising:

(a) a light chain variable region comprising:

(i) CDR-L1 comprising the amino acid sequence SGSSSNIGNNYVS (SEQ ID NO: 41);

(ii) CDR-L2 comprising the amino acid sequence DNNKRPS (SEQ ID NO:42); and

(iii) CDR-L3 comprising the amino acid sequence GTWDSSLTGYV (SEQ ID NO: 43); and

(b) a heavy chain variable region comprising:

(i) CDR-H1 comprising the amino acid sequence SYAIS (SEQ ID NO:44);

(ii) CDR-H2 comprising the amino acid sequence GIIPIFGTANYAQKFQG (SEQ ID NO: 45); and

(iii) CDR-H3 comprising the amino acid sequence YYDFWSGYPGGLFDV (SEQ ID NO: 46).

14 . The isolated antibody or antigen binding fragment thereof of claim 13 , wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:47 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:48.

15 . An isolated antibody or antigen binding fragment thereof, comprising:

(a) a light chain variable region comprising:

(i) CDR-L1 comprising the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 81);

(ii) CDR-L2 comprising the amino acid sequence RNNQRPS (SEQ ID NO:82); and

(iii) CDR-L3 comprising the amino acid sequence AAWDDSLSGWV (SEQ ID NO: 83);

and

(b) a heavy chain variable region comprising:

(i) CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO:84);

(ii) CDR-H2 comprising the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO: 85); and

(iii) CDR-H3 comprising the amino acid sequence SFGPYAFDV (SEQ ID NO:86).

16 . The isolated antibody or antigen binding fragment thereof of claim 15 , wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:87 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:88.

17 . An isolated antibody or antigen binding fragment thereof, comprising:

(a) a light chain variable region comprising:

(i) CDR-L1 comprising the amino acid sequence SGSSSNIGNNYVS (SEQ ID NO: 49);

(ii) CDR-L2 comprising the amino acid sequence DNNKRPS (SEQ ID NO:50); and

(iii) CDR-L3 comprising the amino acid sequence GTWDSSLTGWV (SEQ ID NO: 51);

and

(b) a heavy chain variable region comprising:

(i) CDR-H1 comprising the amino acid sequence SYAIS (SEQ ID NO:52);

(ii) CDR-H2 comprising the amino acid sequence GIIPIFGTANYAQKFQG (SEQ ID NO: 53); and

(iii) CDR-H3 comprising the amino acid sequence YYDFWSGYPGGLFDV (SEQ ID NO: 54).

18 . The isolated antibody or antigen binding fragment thereof of claim 17 , wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:55 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:56.

19 . An isolated antibody or antigen binding fragment thereof, comprising:

(a) a light chain variable region comprising:

(i) CDR-L1 comprising the amino acid sequence QGDSLRSYYAS (SEQ ID NO:57);

(ii) CDR-L2 comprising the amino acid sequence GKNNRPS (SEQ ID NO:58); and

(iii) CDR-L3 comprising the amino acid sequence NSRDSSGNHWV (SEQ ID NO: 59);

and

(b) a heavy chain variable region comprising:

(i) CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO:60);

(ii) CDR-H2 comprising the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO: 61); and

(iii) CDR-H3 comprising the amino acid sequence TNNYGYRYFDY (SEQ ID NO: 62).

20 . The isolated antibody or antigen binding fragment of claim 19 , wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:63 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:64.

21 . An isolated antibody or antigen binding fragment thereof, comprising:

(a) a light chain variable region comprising:

(i) CDR-L1 comprising the amino acid sequence QGDSLRSYYAS (SEQ ID NO:65);

(ii) CDR-L2 comprising the amino acid sequence GKNNRPS (SEQ ID NO:66); and

(iii) CDR-L3 comprising the amino acid sequence NSRDSTDNHLWV (SEQ ID NO: 67); and

(b) a heavy chain variable region comprising:

(i) CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO:68);

(ii) CDR-H2 comprising the amino acid sequence SISSSSSYIYYADSVKG (SEQ ID NO: 69); and

(iii) CDR-H3 comprising the amino acid sequence ATSSGYYYFDY (SEQ ID NO: 70).

22 . The isolated antibody or antigen binding fragment thereof of claim 21 , wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:71 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:72.

23 . An isolated antibody or antigen binding fragment thereof, comprising:

(a) a light chain variable region comprising:

(i) CDR-L1 comprising the amino acid sequence SGSSSNIGNNYVS (SEQ ID NO: 73);

(ii) CDR-L2 comprising the amino acid sequence DNNKRPS (SEQ ID NO:74); and

(iii) CDR-L3 comprising the amino acid sequence GTWDNSLSVWV (SEQ ID NO: 75); and

(b) a heavy chain variable region comprising:

(i) CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO:76);

(ii) CDR-H2 comprising the amino acid sequence YISSSSSTIYYADSVKG (SEQ ID NO: 77); and

(iii) CDR-H3 comprising the amino acid sequence GKGIVGWGFFGMDV (SEQ ID NO: 78).

24 . The isolated antibody or antigen binding fragment thereof of claim 23 , wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:79 and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:80.

25 . A method for detecting KRas-GDP in a biological sample, the method comprising contacting the biological sample with an antibody or antigen binding fragment thereof of any one of claims 1, 5 and 23 .

26 . The method of claim 25 , further comprising contacting the biological sample with an antibody that binds to KRas-GTP, wherein the amount of KRas-GDP and the amount of KRas-GTP are determined.

27 . A method for detecting KRas-GTP in a biological sample, the method comprising contacting the biological sample with an antibody or antigen binding fragment thereof of any one of claims 1, 5 and 23 .

28 . The method of claim 27 , further comprising contacting the biological sample with an antibody that binds to KRas-GDP, wherein the amount of KRas-GTP and the amount of KRas-GDP are determined.

29 . A method of obtaining an inhibitor of a KRas mutant, the method comprising:

contacting the antibody or antigen binding fragment thereof of any one of claims 1, 5 and 23 with the KRas mutant,

screening compounds, and

identifying compounds that bind to the KRas mutant bound to the antibody or antigen binding fragment thereof.

30 . The method of claim 29 , wherein the compounds comprise molecules that covalently modify KRas at the SWII pocket.

31 . The method of claim 29 , wherein the compounds comprise a covalent inhibitor that alkylates at least one residue in the SWII pocket.

32 . The method of claim 29 , wherein the compounds comprise molecules that non-covalently modify KRas at the SWII pocket.

33 . The method of claim 29 , wherein the KRas mutant is KRas G12C , KRas G12V , KRas G12D , KRas G13D , KRas G12R or KRas Q61H .

34 . A method of detecting alkylation of KRas, the method comprising:

contacting a biological sample with the antibody or antigen binding fragment thereof of any one of claims 1, 5 and 23 ; and

detecting the antibody or antigen binding fragment thereof bound to alkylated KRas.

35 . The method of claim 34 , wherein the KRas is KRas G12C .

36 . The method of claim 34 , wherein the antibody or antigen binding fragment thereof is a KRas alkylated conformation specific antibody.

37 . A method of detecting alkylation of KRas in a patient, the method comprising:

administering the antibody or antigen binding fragment thereof of any one of claims 1, 5 and 23 to the patient; and

detecting the antibody or antigen binding fragment thereof bound to the alkylated KRas.

38 . A method of detecting alkylation of KRas in a patient treated with a KRas inhibitor, the method comprising:

(a) obtaining a sample from the patient;

(b) contacting the sample with the antibody or antigen binding fragment thereof of any one of claims 1, 5 and 23 ; and

(c) measuring an amount of KRas bound by the antibody or antigen binding fragment thereof.

39 . The method of claim 38 , wherein the KRas inhibitor is MRTX849, AMG-510, GDC-6036, ARS-3248, LY3499446, LY3537982, or JNJ-74699157.

40 . The method of claim 38 , wherein the amount of KRas bound by the antibody or antigen binding fragment thereof determines a dosage of the KRas inhibitor to administer to the patient.

41 . The method of claim 37 , wherein the KRas is KRas G12C .

42 . The method of claim 37 , wherein the antibody or antigen binding fragment thereof is a KRas alkylated conformation specific antibody.

43 . A method of treating a KRas G12C mediated cancer, the method comprising:

administering, to a patient having a KRas G12C mediated cancer, the antibody or antigen binding fragment thereof of any one of claims 1, 5 and 23 .

44 . The method of claim 43 , wherein the KRas G12C mediated cancer is NSCLC, colon cancer, or pancreatic cancer.

45 . A method for crystallizing KRas, wherein the KRas is optionally bound to a KRas inhibitor, the method comprising:

contacting the antibody or antigen binding fragment thereof of any one of claims 1, 5 and 23 with Kras; and

resolving a crystal structure of the complex.

46 . The method of claim 45 , wherein the KRas is KRas G12C , KRas G12D , KRas G12V , KRas G12R , KRas G13D , or KRas Q61H .

47 . A system for measuring binding of an inhibitor compound to a KRas, the system comprising:

(a) a sensor chip; and

(b) a biosensing surface attached to the sensor chip;

wherein the biosensing surface comprises:

a hydrogel into which a KRas protein and the antibody or antigen binding fragment thereof of any one of claims 1, 2, 5, 6, 23 and 24 are co-localized, wherein:

the KRas protein and the antibody or antigen binding fragment thereof have sufficient degrees of freedom within the hydrogel to engage each other to form affinity complexes;

the local concentration of the KRas and the antibody or antigen binding fragment thereof exceeds the dissociation affinity constant by at least 10-fold, wherein the local concentration promotes formation of the affinity complexes; and

the fraction of unbound KRas protein and anti-KRas antibody is less than about 50%; and

wherein the sensor chip comprises at least one sensing channel for measuring binding of the inhibitor compound to the anti-KRas antibody, wherein the inhibitor compound has been injected onto the biosensing surface.

48 . The system of claim 47 , wherein the hydrogel is about 10-500 nm, 10-300 nm, 10-250 nm, or about 10-200 nm in thickness.

49 . The system of claim 47 , wherein KRas is biotinylated.

50 . A method of measuring binding of a KRas mutant protein to an anti-KRas antibody, wherein the method comprises:

(i) contacting the KRas mutant protein with a biosensing surface to form a KRas-bound biosensing surface;

(ii) contacting the KRas-bound biosensing surface with the antibody or antigen binding fragment thereof of any one of claims 1, 5, and 23 , wherein the antibody or antigen binding fragment thereof is at a molar excess compared to the KRas mutant protein; and

(iii) detecting the binding affinity of the antibody or antigen binding fragment thereof to the KRas mutant protein using surface plasmon resonance,

wherein the biosensing surface is attached to a sensor chip, and

wherein the biosensing surface comprises a hydrogel into which a KRas mutant protein and the antibody or antigen binding fragment thereof are co-localized.